Production of soluble keratin derivaties

ABSTRACT

A process for the preparation of soluble proteins of high molecular weight with little or no damage to the structural integrity of the proteins. The process is economically and environmentally acceptable by virtue of the cost of reagents that are used, and the recycling of some of those reagents, and is suitable for the production of soluble proteins on a large scale. The process includes a first stage using oxidative sulfitolysis followed by a second stage using mild conditions to extract the soluble protein. In the case of wool as the protein source the process leads to the production of soluble keratin proteins fractionated into the classes S-sulfonated keratin intermediate filament proteins and S-sulfonated keratin high sulfur proteins.

FIELD OF THE INVENTION

This invention relates to a process for the preparation of derivativesof keratin from animal sources such as wool, hair, horns, hooves,feathers and scales by an economic and environmentally acceptableprocess, and to a series of keratin derivative products producedthereby. Some of the keratin derivatives are soluble and can be used inthe production of a range of biopolymer materials.

BACKGROUND OF THE INVENTION

Keratins are a class of structural proteins widely represented inbiological structures, especially in epithelial tissues of highervertebrates. Keratins may be divided into two major classes, the softkeratins (occurring in skin and a few other tissues) and the hardkeratins, forming the material of nails, claws, hair, horn, feathers andscales.

The toughness and insolubility of hard keratins, which allow them toperform a fundamental structural role in many biological systems, arealso desirable characteristics in many of the industrial and consumermaterials currently derived from synthetic polymers. In addition topossessing excellent physical properties, keratin, as a protein, is amaterial with a high degree of chemical functionality and, consequently,exhibits many properties that synthetic materials cannot achieve.Keratin is, therefore, well suited to the development of products withhigh-value, niche-market applications. Keratin is also anenvironmentally acceptable polymer produced from a sustainable resourceand therefore has environmental benefits over synthetic materials.Following the global trend of developing materials from renewablesources produced in a sustainable process, a range of materials has beenproduced from keratin, most commonly in the form of keratin films.

At the core of a new industry producing biopolymer materials fromkeratin it is essential to have a process for extracting keratin fromits source that is economically viable, sustainable from anenvironmental perspective, and produces a stable and versatile product.Methods used to date for the extraction of keratin that maintain theintegrity of the individual proteins have been designed for the purposeof protein analysis and characterisation and consequently are not viableon an industrial scale, from an economic and environmental viewpoint.Methods used to date for the economic dissolution of keratin havesignificantly degrading effects on the protein, and consequently thedissolved protein retains few of the physicochemical properties thatlead to the desirability of keratin as a biopolymer, such as the abilityto reconstitute into tough materials.

It is an object of the invention to go some way in overcoming thedisadvantages with known processes or at least provide the public with auseful choice.

In at least one embodiment the invention strives to provide an economicand environmentally acceptable process for the dissolution of keratinproteins that maintains the structural integrity and chemicalfunctionality of the proteins during the dissolution process and leadsto a stable and versatile keratin derivative product for the developmentof biopolymer materials.

SUMMARY OF THE INVENTION

According to a first aspect the invention provides a dissolution processfor producing a range of stable, soluble keratin derivatives of highmolecular weight, the molecular weight being similar to or greater thanthat of proteins originally expressed in the keratin source, with littleor no damage to the structural integrity of the constituent proteins.The dissolution occurs in a two-stage process.

According to a preferred aspect the invention provides a process for thepreparation of keratin derivatives of high molecular weight, the processincluding a first stage digestion step of S-sulfonating a keratin sourceby oxidative sulfitolysis followed by a second stage extraction stepusing controlled washing with water to thereby obtain a highlyS-sulfonated keratin derivative.

The conversion of highly S-sulfonated keratin from a solid state intosolution is without the use of chaotropic agents, by controlled, gradualwashing of the sulfonated keratin with water in order to wash out theresidual chemical reagents from the extraction procedure and alter theionic strength of the extraction solution.

The first stage involves oxidative sulfitolysis to convert cystinegroups present in the protein to S-sulfocysteine, using industriallyacceptable concentrations of inexpensive reagents for the purpose ofsulfonation (eg. sodium sulfite) and oxidation (eg. cupraammoniumhydroxide).

According to another aspect, the invention provides a process for theseparation of a gelatinous keratin product from a solution ofS-sulfonated keratin produced by the above process, wherein theS-sulfonated keratin derivative solution is treated by the use of agentle, gravity fed filtration system followed by separation. Preferablythe separation is centrifugal.

According to another aspect of the invention, a liquid stream remainingafter the gelatinous keratin is removed is processed by passing overscoured wool, thereby removing residual chemicals from the solution andpreparing the wool for subsequent protein extraction processes.

Following conversion of the cystine groups, the second stage of theprocess is one in which the highly S-sulfonted keratin derivative isbrought from a solid or gelatinous state into solution by extensivedilution with water. The rate and extent of dissolution can becontrolled by the use of heat, surfactants, gentle agitation andvigorous chopping or homogenisation. By controlling the rate ofdissolution, reaction solutions can be isolated, for example if a copperoxidant is used a reaction solution rich in copper is produced but itcontains little or no dissolved protein, or are rich in protein butcontain little or no copper.

According to another aspect of the invention, a liquid stream resultingfrom the second stage of the process, which contains residual chemicalssuch as copper sulfate and sulfite, as well as S-sulfonated keratinderivatives, is processed using any one or more of a variety of methodsthat allow the recycling of reagents from the solution and the separateisolation of purified S-sulfonated Keratin Intermediate FilamentProtein(s) (SIFP) and S-sulfonated Keratin High Sulfur Protein(s)(SHSP). This is achieved through the use of chelating agents, such asethylenediamine tetraacetic acid, or chelating ion exchange resins, suchas those containing the iminodiacetic functional group, and the use ofisoelectric precipitation to separate protein types. Ultrafiltration canbe used at several stages in the process to improve the efficiency ofreagent removal or protein separation. Metallic impurities in theprotein products can be further reduced by the washing of the proteinderivative(s) following precipitation with dilute acids, water orchelating agents. Once separated, the protein products can be dried by arange of methods such as fluid bed, spray or freeze drying.

Another aspect of the invention is the further processing of residualkeratin not dissolved by the two stage sulfitolysis process, through theuse of other reagents, such as hydrogen peroxide, sodium sulfide orproteolytic enzymes, to produce keratin peptides.

Another aspect of the invention is the provision of a method for largescale recovery of proteins from a natural source, including subjectingsaid natural protein source to a treatment sufficient to render at leastsome of the protein(s) water soluble, and subsequently separating thewater soluble protein(s).

Another aspect of the invention is the provision of an installation forlarge scale recovery of proteins from a natural source, a treatmentvessel to contain and subject a large quantity of natural protein sourceto a treatment sufficient to render at least some of the protein(s)contained in said feed, water soluble, and a separation apparatus tosubsequently separate the water soluble protein(s).

Another aspect of the invention is a method of selectively solubilisinga protein having plurality of disulfide bonds from a mixture of proteinsincluding subjecting said mixture of proteins to oxidative sulfitolysisto produce a soluble S-sulfonated protein fraction. The oxidativesulfitolysis is preferably effected in the absence of chaotropic agentswith little or no damage to the structural integrity of the protein.

Another aspect of the invention is method for obtaining a purifiedprotein from an impure protein source with little or no damage to thestructural integrity of the protein including subjecting said proteinsource to a treatment sufficient to render at least some of theprotein(s) water soluble, and subsequently separating the water solubleprotein(s) in the absence of chaotropic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a protein extraction process diagram and is referred to below.

DESCRIPTION OF PREFERRED EXAMPLES OF THE INVENTION

The combination of aspects that make up the process as a whole aresummarized diagrammatically in attached FIG. 1.

This process method is for the preparation of highly sulfonated keratinderivatives and can be applied to any keratin source, such as animalwool, hair, horns, hooves, feathers or scales. Whilst the application ofthe method to different keratin sources can give soluble keratins withdifferent structure and properties, the fundamental step of thedissolution process, in which cystine is converted to s-sulfocysteine,applies equally well to all keratin-containing materials.

The process can be conceived as occurring in two stages.

Stage one, which involves the conversion of cystine to S-sulfocysteine,occurs through a procedure of oxidative sulfitolysis. This can beachieved by the use of a sulfonating agent, such as sodium sulfite orsodium metabisulfite, which asymmetrically cleaves the cystine tocysteine and S-sulfocysteine, and an oxidant, which converts thecysteine produced in sulfonation to cystine. By further sulfonation ofcystine complete conversion of all cystine to S-sulfocysteine isachieved.

Oxidants which can be used include sodium tetrathionate, iodosobenzoateand cuprammonium hydroxide. In a preferred embodiment of this inventionthe sulfonating reagent used is sodium sulfite in the concentrationrange 0.02M to 0.2M and the oxidant used is cuprammonium hydroxide inthe concentration range 0.02M to 0.08M, generated by the combination ofcopper sulfate and ammonia. The first stage of the procedure forsolublising keratin is the soaking, for a residence time such as 24hours, of the keratin source in a solution or sequence of solutions thatconvert the cystine to S-sulfocysteine, with a liquor to wool ratio(volume:weight) in the range 5:1 to 50:1.

In another embodiment of the invention the sulfonating agent used issodium metabisulfite in the concentration range 0.1 M to 0.5M,maintained at acidic pH. In this embodiment the wool is removed from thesolution containing sodium metabisulfite before being added to asolution containing a cuprammonium complex in the concentration range0.02M to 0.08M.

Previous work relating to the use of the oxidative sulfitolysisprocedure has required the use of large concentrations of chaotropicagents, such as urea or guanidinium hydrochloride, in order to swell thekeratin source and facilitate the dissolution of keratin. This procedureis both expensive and impractical on an industrial scale. Previous workrelating to the use of oxidative sulfitolysis using copper as theoxidant has been conducted under conditions of temperature and pH thatare detrimental to the integrity of the protein causing high rates ofconversion of cystine to lanthionine.

Stage two of the process involves the conversion of highly sulfonatedkeratin from a solid state into solution without the use of chaotropicagents and under conditions of temperature and pH that maintain thestructural integrity of the protein, by controlled, gradual washing ofthe sulfonated keratin with water in order to wash out the residualchemical reagents from the extraction procedure and alter the ionicstrength of the extraction solution. This combination of effects resultsin the conversion of the highly sulfonated keratin from the solid stateinto aqueous solution. In the preferred procedure the reaction volume isreplaced every 12 to 48 hours, either in a batch process or on acontinuous basis.

The rate and extent of dissolution can be controlled by the use ofsurfactants, the action of heat, agitation, and homogenisation of thesulfonated keratin. A feature of the invention is to use these factorsto control the rate of extraction. The highly S-sulfonated keratin can,therefore, be kept in the solid state and separated from the extractionsolution containing the bulk of the chemicals used for the sulfonationprocess. The preferred procedure uses a non-ionic surfactant, such asTriton X 100 in the range 0.1% to 5% by weight, and a temperaturemaintained in the range 15° C. to 50° C.

An advantage of the invention when a copper based oxidant is used is there-use of this copper-rich extraction solution for subsequent extractionprocesses, significantly reducing both the cost and environmental impactof the process. Re-use of the copper-rich solution is possible due, inpart, to the regeneration of the active copper species through aerialoxidation. One method in which the copper-rich solution can beefficiently reused is by passing the solution over wool. Wool bindscopper from the solution, and if this wool is then used for subsequentextraction processes, the demand for copper in those subsequentextractions is reduced. In this way, a ‘wool filter’ can be used as akey step in the processing of the copper-rich extraction solution,reducing the subsequent need for effluent treatment and also the needfor copper to be added to the subsequent processes. In a typicalprocedure the liquid stream from stage 1 contained approximately1800–1500 (parts per million) ppm copper, and after passing over thewool filter this was reduced to approximately 400–300ppm.

The first stage of the process, and the recovery of reagents for use inthe process are indicated in the attached FIG. 1.

After S-sulfonation and homogenisation the keratin material becomes agelatinous swollen fibrous mass.

A further advantage of the invention is the separation of the highlyS-sulfonated keratin derivatives in the solid state from solutionscontaining either chemicals used in the extraction process or thekeratin protein in solution. This separation is effectively achieved bythe use of a gentle, gravity based filtration through a fine meshscreen, followed by centrifugal separation of the filtrate from fineparticulates.

Solutions of highly S-sulfonated keratin derivatives can be purifiedwith regard to metal ions, specifically the copper ions used as part ofthe extraction process, through the use of ion exchange media, inparticular those containing iminodiacetic acid functionality known topossess a high affinity for divalent metal ions. This ion exchangemedium may be present in the form of a packed resin column, over whichthe protein solution is passed, or it may alternatively form part of anelectrochemical cell, in which copper is recovered from the ion exchangemedium through the use of an applied voltage and a system containingpermeable membranes.

Once the highly S-sulfonated keratin derivates are in solutionparticular proteins eg. the S-sulfonated keratin intermediate filamentprotein can be readily isolated by isoelectric precipitation, around pH4 or below, using acids such as sulfuric acid, hydrochloric acid, citricacid or acetic acid, with the preferred procedure using sulfuric acid.An advantage of the invention is the minimisation of the binding ofcopper and other metallic impurities to the protein prior to isoelectricprecipitation through the use of ion exchange media as described, or byaddition of a chelating agent, such as ethylenediaminetetraacetic acid(EDTA), to the protein solution. In the preferred example EDTA (0.2M) isadded to the liquid stream from stage 2 at a rate of 25 ml per liter, orat a rate suitable to sequester all the copper ions present in solutionas indicated by analysis of the solution. Metallic impurities can befurther reduced by the washing of the protein, once isolated byprecipitation, with a dilute acid solution, or solution of a chelatingagent such as EDTA, or water.

Following precipitation and washing the separated protein can beisolated in a stable, dry state using drying methods involving air flowsat about ambient temperature, for example with the use of a fluid beddryer. Alternatively, the product can be dried using a freeze dryer. Thedry protein product contains cystine groups in the form of S-sulfonicacid and consequently the protein is only soluble in the presence of abase, such as sodium hydroxide or ammonium hydroxide. These processesare represented as drying in the attached FIG. 1.

The highly soluble keratin derivatives that remain in solution followingisoelectric precipitation, which in the case of wool are mainly the highsulfur matrix proteins from within the wool fibre, can be isolated in astable form from solution through a process of ultrafiltration, toremove non-proteinacious species such as residual copper or EDTA,followed by spray drying.

A feature of the invention is the use of a combination of isoelectricprecipitation and ultrafiltration followed by spray drying to separatehighly S-sulfonated keratins according to their properties in solution.In the case of wool keratin, this effectively separates the low sulfurintermediate filament protein class from the high sulfur matrix proteinclass and provides two product streams with different chemicalproperties.

A feature of the invention is the preparation of a stable, water solubleform of the highly S-sulfonated keratin derivative, by dissolving theS-sulfonic acid form of keratin in the presence of base and spray dryingthe resulting solution.

A feature of the invention is the combination of engineering componentsto allow solublisation of the keratin and isolation of the S-sulfonatedkeratin from solution in a continuous, semi-continuous, or batchprocess. This combination of engineering components and unit operationsis detailed in FIG. 1.

An advantage of the invention is the recovery and reuse of copper fromthe reaction mixtures and effluent streams of the process. Copper can berecovered using electrochemical methods, including the use of selectivepermeable membranes in order to separate copper ions from EDTA prior toelectrochemical deposition. Alternatively, immobilized binding agents,in the form of copper-specific ion exchange resins, can be used toremove copper from the effluent stream. Copper removed using thesemethods can be reused, thereby minimizing the environmental impact ofthe process.

The use of ion exchange media and/or chelating agents is represented aspurification in the attached FIG. 1.

Another advantage of the invention is the further processing of residualkeratin which remains in the solid state following the extractionprocedure. This functionalised keratin is highly S-sulfonated, thereforethe disulfide bonds present in the native keratin that render itresistant to chemical and enzymatic attack have been cleaved and thekeratin is readily digestible using other extraction methods. Forexample, a solution rich in keratin peptides can be prepared through theaction on this residual keratin of alkaline solutions of a strongoxidant such as hydrogen peroxide, in the concentration range 10–100 mlof 50% hydrogen peroxide per kg of the keratin residue under alkalineconditions. The keratin residue contains approximately 5% solids.Alternatively, solutions of strong reductants such as sodium sulphide inthe concentration range 0.5%–15% added to the keratin residue can beused to prepare a solution rich in keratin peptides. Alternatively,proteolytic enzymes, such as those of the subtilisin, papain or trypsingroups, can be employed at levels in the range 0.1 mg–20 mg of enzymeper gram of keratin residue at temperature and pH conditions appropriatefor the specific enzyme to readily digest the residual keratin andprepare a solution rich in keratin peptides. All of these methods resultin the formation of a solution rich in keratin peptides which can beprocessed in a similar manner to the liquid stream resulting from stage2 described above, that is through the use of ion exchange media, pHadjustment and drying (shown as purification and drying in FIG. 1), toproduce keratin peptide solids. Digestion of the keratin residue in thisway minimises the keratin waste produced by the process as a whole, andensures maximum utility of the keratin protein present in the keratinsource.

The two intact protein products from the process are S-sulfonatedkeratin intermediate filament protein and S-sulfonated keratin highsulfur protein. The S-sulfonated keratin intermediate filament proteintypically produced by the process was analysed using sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysisusing a reduction/alkylation procedure, which indicated a molecularweight distribution predominantly in the range 30–60 kD (intermediatefilament proteins), with a small component of protein of mass 10 kD(high glycine high tyrosine proteins). The amino acid composition ofthis product is given in Table 1 and is typical for wool keratinintermediate filament proteins. The S-sulfonated keratin high sulfurprotein was analysed using SDS-PAGE after a reduction/alkylationprocedure, which indicated a molecular weight predominantly in the range15–20 kD. The amino acid composition of this product is given in Table 1and is typical for wool keratin high sulfur proteins.

TABLE 1 amino acid composition of S-sulfonated keratin intermediatefilament protein (STEP), S-suffonated keratin high sulfur protein(SHSP), intermediate filament protein (IFP) and high sulfur protein(HSP) (later two courtesy of Gillespie and Marshall, Variability in theproteins of wool and hair, Proc. Sixth Int. Wool Text. Res. Conf,Pretoria, 2, 67–77, 1980). All residues expressed as mol %.S-sulfocysteine, cystine and cysteine are measured as S-carboxymethylcysteine following reduction and alkylation. Cya Asp Glu Ser Gly His ArgThr Ala Pro Tyr Val Met Lan Ile Leu Phe Lys Cys SIFP 0.4 7.9 15.4 10.98.1 0.9 7.9 6.5 7.5 5.4 1.1 6.5 0.2 0.2 3.7 8.9 2.5 2.1 4.2 SHSP 1.7 2.68.6 14.3 9.1 0.8 6.8 10.4 3.6 12.6 1.8 6.3 0.0 0.2 2.9 3.9 1.5 0.4 12.4IFP 0.0 9.6 16.9 8.1 5.2 0.6 7.9 4.8 7.7 3.3 2.7 6.4 0.6 0.0 3.8 10.22.0 4.1 6.0 HSP 0.0 2.3 7.9 13.2 6.2 0.7 6.2 10.2 2.9 12.6 2.1 5.3 0.00.0 2.6 3.4 1.6 0.6 22.1

An example of the process is shown diagrammatically in the attachedFIG. 1. Ultrafiltration is considered as being a possible component ineach purification stage. The key components are illustrated by thefollowing examples of a protein extraction procedure.

EXAMPLES Example 1 Stage 1, Digestion

The digestion stage of the process involves the use of oxidativesulfitolysis to convert cystine to S-sulfocysteine within the keratinsource.

Example 1a Stage 1, Digestion

In order to extract the keratin from 10 kg of wool, firstly 2 kg ofcopper sulfate pentahydrate was mixed using a high shear mixer witheight litres of concentrated ammonia. This mixture was diluted to 200 Lwith water and 10 kg of wool was added. Approximately 15 L of sulfuricacid (2M) was-added to the stirred mixture till a pH 9.4 was achieved.Anhydrous sodium sulfite (5.04 kg) was added and the solution mixeduntil complete dissolution of all of the reagents had occurred and thepH stabilised at 9.5. The final concentration of the cupric ammoniacomplex was 0.04M. The sodium sulfite had a final concentration of 0.2M.The temperature of the digestion solution was maintained at 20° C. After24 hours of gentle agitation the fibrous gelatinous mass of softenedwool was filtered. The filtrate was passed through a fresh wool filter,which decreased the copper level in the solution from 1725 ppm to 130ppm, and further purified using Purolite S930 IDA ion exchange resin,which under acidic conditions further reduced the copper level to 12ppm. Fresh water was added to the softened wool and the mixture wasagitated.

Example 1b Stage 1, Digestion with the Use of Surfactant

In a variation of example 1a, the digestion solution was prepared withthe addition of 1% of a non-ionic surfactant Triton X 100. The additionof this surfactant resulted in a delay in the release of soluble proteinfrom the fibre, allowing a more effective separation of protein fromresidual reagents such as copper salts in the extraction solution.

Example 1c Stage 1, Digestion

In a variation of example 1a, the digestion stage occurs in two parts.In the first part wool is pretreated with sodium metabisulfite at aconcentration of 0.2M, at pH 4.2. Following removal of the wool fromthis solution, and with no attempt to remove residual sulfite from thewool, the wool was immersed in a cuprammonium hydroxide solution, at theconcentration and pH described in example 1a for a further 24 hours at20° C.

Example 2 Stage 2, Extraction Example 2a Stage 2, Batch Extraction

Following completion of stage 1, described in examples 1, the mixturewas agitated for a period of 16 hours, before being homogenized.Following a further 4 hours of agitation the solids and solution wereseparated using a two-stage filtration process involving a wedge wirescreen followed by a settling tank and a spinning disc centrifuge. Thesolid phases were returned to the reaction vessel and water was added togive a final liquor to wool ratio of 20:1 based on original wool solids.Following 24 hours agitation or continuous homogenisation the mixturewas separated by repeating the two-stage filtration process. The solidphases were returned to the extraction vessel and further diluted. Thiscycle was repeated 7 to 12 times. The liquid phases, containing solubleproteins, were further processed as detailed below in example 3.

Example 2b Stage 2, Continuous Extraction

Following completion of stage 1 the mixture was processed as describedin example 2a, except that the two stage filtration process occurred ona continuous process, and solids and fresh water were added to thereaction tank at a rate equivalent to the volume of the tank beingreplaced in 24 hours. This process was continued for 120 hours.

Example 3 Processing of Protein Solutions

Ultrafiltration can be used at several points during the processing ofprotein solutions, in order to concentrate solutions and make theprocesses of drying and ion exchange more efficient. Ultrafiltration maybe used prior to any processing step outlined in the following examples.

Example 3a Processing of Protein Solutions Using EDTA

The solution produced as a result of stage 2, as described in Example 2,was further processed to isolate purified soluble keratins. EDTA (0.2M )was added to the liquid phase at a rate of 25 mL per liter, or at a ratesuitable to sequester all the copper ions present in solution asindicated by analysis of the solution. Following 1 hour of mixing, thepH of the filtrate was reduced to 3.5 using sulfuric acid. The proteinprecipitate was isolated using a screen, and washed sequentially withdilute sulfuric acid and water. The protein, S-sulfonated keratinintermediate filament protein, was dried by one of three routes, freezedrying, fluid bed drying or spray drying following dissolution withdilute sodium hydroxide. The filtrate following the proteinprecipitation procedure was further processed using ultrafitration, toseparate the protein components from the residual reagents. Theretentate was spray dried to isolate further soluble protein,S-sulfonated keratin high sulfur protein. The permeate was furtherprocessed to recover copper and EDTA from the effluent stream using ionexchange media.

Example 3b Processing of Protein Solution Using Ion Exchange Media

The solution produced as a result of stage 2, as described in Example 2,was further processed to isolate purified soluble keratins. The liquidphase was passed over ion exchange resin, such as the chelating resinPurolite S930 IDA ion exchange resin containing the iminodiacetic acidfunctional group, in order to remove copper ions from the solution.Following ion exchange the pH of the filtrate was reduced to 3.5 usingsulfuric acid and further processed in an identical manner to thatdescribed for Example 3a.

3c. Processing of Protein Solution Using pH Adjustment Prior to IonExchange.

The solution produced as a result of stage 2, as described in example 2,was further processed to isolate purified soluble keratins. The pH ofthe liquid phase was reduced to 3.5 using sulfuric acid. The proteinprecipitate was isolated using a screen, redissolved using dilute sodiumhydroxide and further purified with either the addition of EDTA or bypassing over an ion exchange column. Following further purification, thepH of the solution was reduced to 3.5 using sulfuric acid and theprotein was isolated as described in the earlier examples. The filtratefrom the initial pH reduction step, which still contains significantamounts of soluble protein and other reagents, was purified by passingover ion exchange media and spray dried to isolate further solubleprotein, S-sulfonated keratin high sulfur protein.

Example 4 Dissolution of Residues from Stage 2

The solid stream isolated as a result of stage 2 can be furtherprocessed to produce keratin peptides by a range of methods. The highlevel of sulfonation of the residue makes it readily amenable tochemical and enzymatic digestion, as the disulfide bonds present in theoriginal keratin source resistive to chemical and enzymatic attack havelargely been cleaved.

Example 4a Dissolution of Residues Using Sodium Sulfide

Sodium sulfide solution (5% by weight) is added to an equal volume ofthe solid stream from stage 2 of the process, which comprisesapproximately 5% solids. The mixture is agitated for 12 hours afterwhich time the solids are removed by filtering and centrifugation andsulfuric acid is added to the protein solution to decrease the pH to therange 2 to 3.5. The precipitate is collected on a screen and washedthoroughly with water.

Example 4b Dissolution of Residues Using Hydrogen Peroxide

Hydrogen peroxide (50%) is added to the solid stream from stage 2 at alevel of 25–30 ml per kg of keratin residue (keratin residue containsapproximately 5% solids). This is mixed and 1 M sodium hydroxide isadded to obtain pH in the range of 10 to 13. The mixture is agitatedgently for 24 hours and the protein and solids separated by the twostage filtration process described in Example 2 and protein isolated byacidification as described in Example 4a. Alternatively the proteinsolution is passed over an ion exchange resin, then acidified and theprecipitated solid collected. The acidified solution may then be passedthrough an ion exchange column prior to freeze-drying or spray drying tocollect a further protein-rich product.

4c Dissolution of Residues Using Proteolytic Enzymes

An industrial subtilisin enzyme preparation (a solution containing 2.5%active enzyme) was added to the solid stream from stage 2 in the amountof 10 mg of active enzyme per gram of keratin residue. The pH wasmaintained at 9.5 with the addition of sodium hydroxide and the reactionheated to 60° C. for 2 hours. The resulting protein solution is isolatedfrom solids and processed as described in 4a or passed throughion-exchange resin prior to and/or following acidification as describedin 4b.

Thus by the invention there is provided a method for the production ofsoluble keratin derivatives that is both economic and environmentallyacceptable.

Particular examples of the invention have been described and it isenvisaged that improvements and modifications can take place withoutdeparting from the scope of the attached claims.

1. A process for preparing S-sulfonated keratin protein comprising thesteps of: (a) reacting a keratin source with oxidative sulfitolysis toproduce solids comprising reaction product in digestion liquor, (b)separating the solids from the digestion liquor, (c) extracting thesolids with a composition consisting of water or water and surfactantand separating extract from solids, where the extraction can berepetitive or continuous, and (d) processing extract to recoverS-sulfonated keratin protein.
 2. The process of claim 1 where thekeratin source is wool.
 3. The process of claim 1 where the post-extractsolids of step (c) comprise water insoluble gelatinous composition. 4.The process of claim 1 where the digestion liquor includes coppercontaining reactant.
 5. The process of claim 4 where the oxidativesulfitolysis provides metal ions which remain in the extract and theprocessing of step (d) comprises sequestering the metal ions in theextract with a chelating agent.
 6. The process of claim 5 where thechelating agent is EDTA.
 7. The process of claim 1, where step (b) isperformed by filtration.
 8. The process of claim 1, where the separationof extract from solids in step (c) is performed by filtration.
 9. Theprocess of claim 1, where oxidative sulfitolysis provides metal ionswhich remain in the extract and the S-sulfonated keratin proteincomprises S-sulfonated high sulfur keratin protein and S-sulfonatedintermediate filament keratin protein.
 10. The process of claim 1, wherestep (a) is performed without the use of a chaotropic agent.
 11. Theprocess of claim 1, where step (a) is performed under conditions of pHthat maintain the integrity of the protein.
 12. The process of claim 1,where the keratin source is wool.
 13. A process for preparingS-sulfonated high sulfur keratin protein from S-sulfonated keratinprotein comprising the steps of: (a) reacting a keratin source withmetal ion containing oxidative sulfitolysis to produce solids comprisingreaction product in digestion liquor, wherein said metal ions remain inthe extract, (b) separating the solids from the digestion liquor, (c)extracting the solids, either repetitively or continuously, with acomposition consisting of water or water and surfactant and separatingthe extract from the solids, wherein said solids comprise a waterinsoluble gelatinous composition, (d) processing the extract to recoverthe soluble keratin proteins, (e) adding acid and water to the extractto precipitate the S-sulfonated keratin intermediate filaments protein(f) filtering the precipitate to produce a filtrate containing theS-sulfonated high sulfur keratin protein, (g) ultrafiltering thefiltrate and recovering the retentate, (h) drying the retentate toproduce purified S-sulfonated high sulfur keratin protein.
 14. A processfor preparing S-sulfonated intermediate filament keratin protein fromS-sulfonated keratin protein comprising the steps of: (a) reacting akeratin source with metal ion containing oxidative sulfitolysis toproduce solids comprising reaction product in digestion liquor, whereinsaid metal ions remain in the extract, (b) separating the solids fromthe digestion liquor, (c) extracting the solids, either repetitively orcontinuously, with a composition consisting of water or water andsurfactant and separating the extract from the solids, wherein saidsolids comprise a water insoluble gelatinous composition, (d) processingthe extract to recover the soluble keratin proteins, (e) adding acid andwater to the extract to precipitate the S-sulfonated intermediatefilament keratin protein, (f) filtering the S-sulfonated intermediatefilament keratin protein and recovering the solids, (g) drying thesolids to produce purified S-sulfonated intermediate filament keratinprotein.
 15. A process for preparing soluble keratin peptides fromS-sulfonated keratin protein comprising the steps of: (a) reacting akeratin source with oxidative sulfitolysis to produce solids comprisingreaction product in digestion liquor, (b) separating the solids from thedigestion liquor, (c) extracting the solids, either repetitively orcontinuously, with a composition consisting of water or water andsurfactant and separating the extract from the solids, wherein saidsolids comprise a water insoluble gelatinous composition, (d) contactingthe insoluble gelatinous composition of (c) with an agent selected fromthe group consisting of sodium sulfide solution, hydrogen peroxidesolution in the presence of residual metal ions and proteolytic enzymesto produce soluble keratin peptides containing solution, and (e)processing the solution of (d) to recover the soluble keratin peptides.16. The process of claim 15, wherein the proteolytic enzyme is from thesubtilisin, papain or trypsin family.